From 2010.igem.org
Building Parts
10-05-2010
PCR a Kanamycin resistance cassette fragment from p1003 with primers containing either the A/B' ends or the B/A' ends.
Recipe:
- 1uL p1003 (approx. 1ng)
- 2.5uL prA_p1003+
- 2.5uL prB'_p1003-
- 5uL 10X PCR buffer
- 1uL 10uM dNTPs
- 2uL 50uM MgCl~2~
- 0.5uL Taq polymerase
- 35.5uL MilliQ H~2~O
Program:
11-05-2010
June 3, 2009
- Plasmid transformed = pSB1AC3 (TEST)
- Note: team members were of mixed opinion as to whether the transform was likely due to low availability of DNA - DNA was split between a number of transformation reactions for at least 2 different DB3.1 strains. Plates incubated at 30 degrees Celsius overnight, temp. raised to 37 degrees approx. 10:30 a.m.
June 5, 2009
- Tet plates made
- Recipe for 200 ml (approx. 10 plates):
- 2.2 g agar in 200 ml fresh LB
- Note: do not re-autoclave LB, it will caramelize!
- Recipe for 200 ml LB:
- a) 1 g yeast extract
- b) 2 g peptotryptone
- c) 2 g NaCl
- d) 200 ml water
- Autoclaved at 11:20 a.m., picked up at 1:15 p.m. and cooled in water bath at 37 degrees Celsius
- Added 200 microlitres tetracycline stock (from -20 freezer) to final concentration of 20 microgram/ml
- Swirl and poured into prepared, labeled plates
- Note: label on plates reads June 3, 2009 but should be June 5, 2009 (8 plates in total - either I overpoured or these plates are a bit larger than I was used to)
- Inverted and put in 37 degree incubator to dry
June 8, 2009
- Bacteria grown to log phase using sterile inoculating loop to inoculate approx. 200 ml of LB media with bacteria from 2 plates (approx. 3 colonies)
- Bacterial liquid culture placed in shaker at 10:51 a.m.
June 9, 2009
- Digested miniprepped gel with EcoRI and SpeI
- Running gel to see if correct plasmid was purified - should expect to see bands at ~3200 bp
- DNA Ladder made - 6 microlitres of stock used